Summary of Work: The pharmacological and physiological activity of recently developed opioid mimetic substances were determined in vivo using mice in comparion to morphine and using general as well as specific opiate antagonists to assess their mode of action. The antinociception profile paralled that of the in vitro functional pharmacological data using guinea-pig ileum and mouse vas deferens assay systems, and reflected the receptor binding affinity (see previous projects for details on the interaction of peptides with delta- and mu-opioid receptors). A series of novel Dmt-containing pyrazinone opioid mimetic compounds had exceptional activity in vivo, one of which was 60- to 71-fold more potent than morphine in generating analgesia using standard testing procedures after i.c.v. administration, but depending on whether the test measured spinal or supraspinal effects; the former being more potent than the latter. In particular, the compound 3-[4'-Dmt-aminobutyl)-6-(3'-Dmt-aminopropyl0-5-methyl-2(1H)pyrazinone had very high affinity (Ki mu = 0.02 nM), selectivity (delta/mu = 1,520) and agonist activity (GPI, IC50 = 1.7 nM) with weak activity toward the delta receptor in all assay systems. This compound acted to produce spinal antinociception primarily through spinal antinociception using the mu-2 receptor subtype; however, the mu-1 subtype dominates supraspinally. The s.c. injection produced CNS-mediated antinociception, providing evidence the compound passed through memrane barriers in both the gastrointestinal tract and in the microcapillary tight junctions in the brain. However, a tolerance was obtained after a week that was equivalent to morphine that suggests that both substances act through similar mechanisms at mu-opioid receptors. Another class of Dmt-containing pyrazinone compounds, the 3,6-bis-[Dmt-NH-(CH2)n]-2(1H)-pyrazinone compounds not only exhibited central (CNS) mediated analgesia, but also were orally bioavailable opioid mimetics. These symmetric substances displayed high affinity for mu-opioid receptors (Ki = 0.04-0.12 nM) and potent angonism on GPI (IC50 = 1.3-1.9 nM). They produced analgesia in vivo by i.c.v. administration that was 50- to 63-fold more potent than morphine, but only about half as potent when injected s.c. or administered orally, which is still orders of magnitude greater than other endogenous opioid peptides and, importantly, were unmodified by glycosylation, adamantane, triglycerides, halogens, antibody, biotin, bulky organic molecules, esterification of a C-terminal carboxyl group (of which none exist in these compounds) or O-acylation of the phenolic hydroxyal group of Tyr or Dmt, nor was it necessary to absorb them onto polysorbate coated nanoparticles to induced uptake through the BBB.